Bones and skeletal muscles make up approximately 20 and 45%, respectively, of the weight of the human body. They have several vital functions. For example, locomotion, breathing, postural support, physical protection, blood glucose disposal, thermogenesis, Ca2+ homeostasis, production of blood cells, and energy storage are all under the control of bones and skeletal muscles. Musculoskeletal diseases are a major burden on individuals and the health and social care systems, with major indirect costs1. The prevalence of many musculoskeletal problems increases markedly with age, obesity, and lack of physical activity1. These three risk factors are expected to increase steadily over the next decade, putting people at increasingly higher risk for musculoskeletal diseases. The United States Health Examination Survey indicated that 30% of the population aged between 25-74 had musculoskeletal symptoms2. More importantly, in Canada, the estimated number of people with disabling musculoskeletal disorders is more than twice that for all cancers combined3. Clinical studies have shown the worsening of osteoporosis and muscle atrophy/dysfunction occurs in parallel4.
Skeletal muscles and bones remain plastic, work in synchrony, and have the ability to adjust their structures in response to their mechanical, hormonal, and metabolic environments5. This is best exemplified by professional tennis players, whose dominant arm has stronger muscles and greater bone mass. Skeletal muscle and bone atrophy (loss of muscle and bone mass) occur with aging, prolonged bed rest, strokes, spinal cord injuries, burns, neurodegenerative diseases, space flight, immobilization, arthritis, osteoarthritis, denervation, and a number of other debilitating conditions6,7,8,9,10,11,12,13,14,15,16. In addition, long-term glucocorticoid administration (e.g., dexamethasone), which is an anti-inflammatory and immunosuppressant, induces osteoporosis and muscle atrophy/dysfunction17, while local and systemic alterations in hormone and pro-inflammatory cytokine levels stimulate muscle and bone atrophy18,19. Changes in intracellular Ca2+ concentrations also regulate the physiological activities and expression of specific bone and muscle genes20,21. Physical exercise and mechanical stimuli, on the other hand, promote increased bone density and skeletal muscle hypertrophy22,23.
Osteoblasts in bone produce the extracellular matrix, cytokines, and growth factors. They are also involved in the regulation of bone formation and resorption in response to hormonal and local factors. Like macrophages, osteoclasts originate from myeloid cells and play key roles in bone degradation and remodelling. One advance in bone biology and disease was the discovery of the receptor-activator of nuclear factor κβ (RANK), receptor-activator of nuclear factor κβ ligand (RANKL), and osteoprotegerin (OPG) triad (RANK/RANKL/OPG). RANK/RANKL triggers a network of TRAF-mediated kinase cascades that promote osteoclast differentiation. RANKL is expressed on osteoblast cells and its receptor, Rank, on pre-osteoclastic cells. RankL production is stimulated by IL-1, IL-6, IL-11, IL-17, TNF-α, vitamin D, Ca2+, parathyroid, glucocorticoids, prostaglandin E2, and immunosuppressive drugs, and is down-regulated by TGF-α24. The RANK/RANKL interaction induces the differentiation and formation of multinucleated mature osteoclasts, causing bone resorption. The third protagonist, OPG, is also produced by osteoblasts and exerts an inhibitory effect on the pre-osteoclastic differentiation process. OPG, by binding to RankL, inhibits the RANK/RANKL interaction and subsequent osteoclastogenesis. OPG is thus a very efficient anti-resorptive agent. It also serves as a decoy receptor for the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and increases cell survival by blocking the apoptotic effects of this ligand. The fact that the overexpression of OPG in mice results in severe osteoporosis and that OPG-null mice are osteoporotic is testimony to the physiological importance of OPG25,26,27. The lack of RANK or RANKL induces osteoporosis in mice28,29.
Muscle wasting/dysfunction is a hallmark of diverse catabolic conditions, including muscle disuse, burn injuries, cancers, renal failure, AIDS, chronic obstructive pulmonary disease, and aging31,32,33,34. While calpain and the inhibition of the autophagy/lysosome system can induce muscle protein degradation, the ubiquitin/proteasome pathway appears to be the most important system involved in muscle proteolysis35. For example, the ubiquitin ligase muscle atrophy F-box (MAFbx or atrogin-1) and muscle ring finger 1 (MuRF1), which target muscle-specific proteins for degradation by the proteasome, are up-regulated and are two of the genes most affected by various types of muscle atrophy36,37. Conversely, hypertrophy is in part mediated by IGF-1 via the stimulation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway38. In transgenic mice, the over-expression of IGF-1 or the active form of Akt is sufficient to induce skeletal muscle hypertrophy39,40. Akt downstream targeting of glycogen synthase kinase (GSK)-3beta, the mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K), and the phosphorylation of forkhead family transcription factor Forkhead box 0 (FOXO) prevent the transcription and activation of MAFbx and MuRF141,42.
Bone resorption is regulated through the expression of OPG and RANKL by osteoblastic cells and is altered by various osteotropic factors, such as vitamin D, that regulate Ca2+ influx. Vitamin D changes the functional properties of L-type voltage sensitive Ca2+ channels (L-type VSCC) and alters the expression and activity of protein kinases43,44,45. L-type VSCC is the primary site for Ca2+ influx into proliferating osteoblasts48. Once Ca2+ accumulates intracellularly, calmodulin (CaM), a major intracellular Ca2+ receptor, can interact with and regulate various proteins, including Ca2+ channels, Ca2+/calmodulin-dependent protein kinase (CaMK), and calcineurin, all of which can control transcriptional expression46. The transient elevation of intracellular Ca2+ directly or indirectly influences the expression and activity of intracellular protein kinases, including c-AMP dependent protein kinase A (PKA), CaMK, and MAPK45,47, which can potentially phosphorylate L-type VSCC and alter channel function. More importantly, there is a clear feedback loop between OPG and RANKL that serves as a major regulatory mechanism for controlling osteoclastogenesis and L-type VSCC, thus modulating Ca2+ influx into osteoblasts. This is best exemplified by the fact that OPG secretion by osteoblasts is regulated through CaMK signalling, which depends on the activity of L-type VSCC48. L-type VSCC is so important that blocking its function inhibits osteogenesis, produces vertebral defects, and decreases mineral apposition49.
In skeletal muscle, the sequence of events that converts an electrical stimulus (alpha motor neurons and action potential) to a mechanical response (muscle contraction) is defined as excitation:contraction coupling (ECC). This essential sequence of events in muscle physiology involves the depolarization of the transverse-tubular (t) system, which activates dihydropyridine receptors (DHPRs), also called L-type voltage dependent Ca2+ channels, an analogous to L-type VSCC. The activation of DHPRs opens ryanodine receptor/Ca2+ release channels (RYR1) adjacent to the sarcoplasmic reticulum (SR) membrane, resulting in the rapid efflux of large of amounts of Ca2+ into the cytoplasm and the binding of Ca2+ to troponin C and then actin and myosin to form cross bridges, shortening the sarcomere and decreasing force development50. To avoid permanent muscle contraction, Ca2+ is pumped back into the sarcoplasmic reticulum by sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA). Calsequestrin can then bind free Ca2+ in the SR so that SERCA does not have to pump against a high concentration gradient. It is important to mention that the Ca2+ concentration is 10,000 times higher in the SR than in intracellular compartment under basal and resting conditions. The release of Ca2+ by RYR1 and the reuptake of Ca2+ by SERCA are also tightly regulated by several binding proteins. Calstabin1, PKA, and protein phosphatase 1 (PP1) control the open and closed state of the RYR1 channel. PKA mediates the phosphorylation of RYR1 at Ser2844, increases the sensitivity of the channel to cytoplasmic Ca2+, reduces the binding affinity of calstabin1 for the RyR1 complex, and destabilizes the closed state of the channel, leading to Ca2+ leakage51,52. The rate at which SERCA moves Ca2+ across the SR membrane can be controlled by phospholamban under β-adrenergic stimulation. For instance, the movement of Ca2+ is reduced when phospholamban is associated with SERCA while the dissociation of phospholamban increases SERCA activity and Ca2+ movement. From a physiological point of view, SERCA works at sub-maximal levels in resting cardiac and skeletal muscles, which allows intense physical performance (increased muscle force and speed) as needed when phospholamban is phosphorylated and dissociated from SERCA. This phenomenon is tightly linked to the well-known fight or flight response, which is under the control of the sympathetic nervous system (catecholamine hormones; adrenaline and noradrenaline). Under pathological and chronic stress conditions, constant Ca2+ leakage and dysfunctional Ca2+ mobilization impair muscle force development and may activate Ca2+-dependent proteases, including calpain, leading to a detrimental effect on cell viability.
Skeletal muscles are primarily composed of four muscle fibre types: type I fibres (slow and oxidative), type IIa fibres (fast and oxidative), and type IIb fibres (fast and glycolytic). Type I fibres play an important role in maintaining body posture, while type IIb and IIx fibres are responsive during physical activity. Type IIa fibres are a hybrid between type I and type IIb fibres and can perform short or prolonged exercises. Specific muscle diseases, mechanical stress, and drug treatments affect all four muscle fibre phenotypes to different degrees. For example, a decrease in mechanical load and neuromuscular activity favours muscle atrophy and a conversion of muscle fibre phenotypes from slow to fast53. Functional overloads cause a gain in muscle mass while prolonged exercises lead to the transformation of pre-existing fast-twitch muscle fibres to a slow-twitch oxidative phenotype54. Additionally, sarcopenia (progressive loss of skeletal muscle mass and strength during aging) affects oxidative and glycolytic muscle fibres differently. For example, type II muscle fibres begin to atrophy in humans during the fifth decade while type I muscle fibres maintain their size for most of a human's lifetime. Prolonged glucocorticoid treatments mainly affect fast twitch muscle fibres, leaving slow twitch muscle fibres intact. Type IIb fibres are converted to oxidative phenotype fibres (type I or IIa) or disappear first through a necrotic process in mdx mice and DMD patients. The accumulated evidence indicates that type IIb fibres, which are essential for brief and powerful contractions (i.e., standing up from a chair), are the most vulnerable muscle fibres in several types of myopathy.
Proinflammatory cytokines TNF-α and IL-1 activate transcription factor NF-kB, which can abrogate muscle proliferation, differentiation, and growth in several chronic and inflammatory diseases. While there is strong evidence that NF-kB regulates muscle mass, other transcription factors also play an important role in the regulation of muscle mass. In cancer cachexia, myostatin-induced muscle atrophy is regulated through FOXO-1 and the E3 ubiquitin ligase gene MAFBx/atrogin-1, a process that is independent of the NF-kB/MuRF1 mechanism55. Furthermore, sepsis results in a sustained increase in the expression and activity of AP-1 and C/EBP56,57, which are, in part, regulated by glucocorticoids58. Other observations indicate that Ca2+ concentrations and the expression of muscle m-, μ-calpain are important in muscle atrophy and dysfunction in septic muscle59. Furthermore, treating septic rats with dantrolene, a substance that inhibits the release of Ca2+ from intracellular stores, prevents the sepsis-induced release of myofilaments59. Ca2+ also regulates phosphorylation and dephosphorylation by activating CaMK and calcineurin60, leading to an increase in proteasome activity61. Muscle atrophy/dysfunction is thus clearly under the control of several signalling pathways.
There is a need for new therapy for treating neuromuscular disorders, non-genetic myopathies, genetic myopathies and/or for regulating skeletal or cardiac muscle disuse, diseases and aging.